4. RESULTS
4.2. Clinicopathological characteristics of lung adenocarcinoma patients
In order to determine the clinical relevance of KRAS and EGFR mutations, we performed a comparative statistical analysis of mutational status and clinicopathological variables (summarized in Tables 3, 4, 5, 6, and 7). The major clinicopathological characteristics could be collected in cohort #1 and in cohort #2 (505 and 645 patients, respectively) and are presented for the various mutational statuses in Tables 3 and 4. Similarly to the cohort
#1, significant association between gender or ECOG PS and mutational status was not detected in cohort #2 (Tables 4 and 5, and Figure 8A).
In cohort #1, KRAS mutation was not significantly associated with age when patients were grouped as <55, 55-64 and 65≤ years (P=0.119). However, one-way analysis of variance (ANOVA) test with Tukey Multiple Comparison indicated a significant difference between the average ages of WT and KRAS codon 12 mutant patients (60.7 versus 58.8 years, respectively, P=0.032). In cohort #2 patients with KRAS mutations (mean age ±SD, 60±10.4 yrs.) were significantly younger than those with EGFR/KRAS double WT tumors (mean age ±SD, 64±9.7 years) or with classic EGFR mutations (mean age ±SD, 67±9.6 years) (P<0.001, Figure 8B).
We found no significant association with major clinicopathological factors and amino acid-specific KRAS mutation subtypes.
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Table 3. Correlation of clinicopathological features and KRAS mutational status in patients with advanced pulmonary adenocarcinoma in cohort #1 (n=505).
KRAS status
a Mean age was 60.1 years (range, 33-79; SD=8.04) for the entire patient population, 60.7 years (range, 33-79; SD=7.93) for the wild-type (WT) patients, 58.8 years (range, 39-78; SD=8.16) for the KRAS codon 12 mutant group, and 58.1 years (range, 47-73;
SD=8.02) for the KRAS codon 13 mutant cohort. b In 44 cases, smoking status was not available; Data shown in parentheses are column percentages; ECOG PS, Eastern Cooperative Oncology Group performance status
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Table 4. Characteristics of patients with major clinicopathological data available in cohort #2 (n=645).
Data shown in parentheses are column percentages.
* In cohort #2, out of the total number of patients (n=814) with molecular analysis, clinicopathological data was available in 645 cases. EGFR molecular analysis was not done in 43 cases.
ECOG PS, Eastern Cooperative Oncology Group performance status
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Figure 8.Epidemiology of KRAS and EGFR mutations in lung adenocarcinoma patients in cohort #2. (A) There was no significant association between mutational status and gender. (B) Patients with KRAS mutation were significantly younger than those with classic EGFR mutations or with EGFR/KRAS double wild-type (WT) tumors (P<0.001).
Table 5. Correlation of clinicopathological features, and KRAS codon 12 mutation subtypes in cohort #1 in patients with advanced pulmonary adenocarcinoma (n=136 a).
KRAS mutation a G12C (n=61) G12V (n=29) G12D (n=27) Rare (n=19) P value
Age b (years)
<55 15 (24.6%) 6 (20.7%) 7 (25.9%) 4 (21.1%)
0.767 55-64 35 (57.4%) 16 (55.2%) 13 (48.1%) 8 (42.1%)
>65 11 (18%) 7 (24.1%) 7 (25.9%) 7 (36.8%) Gender Male 28 (45.9%) 14 (48.3%) 13 (48.1%) 5 (26.3)
0.407 Female 33 (54.1%) 15 (51.7%) 14 (51.9%) 14 (73.7%)
Smoking Never-smoker 3 (4.9%) 6 (20.7%) 1 (3.7%) 3 (15.8%)
0.055 Ever-smoker 58 (95.1%) 23 (79.3%) 26 (96.3%) 16 (84.2%)
ECOG PS 0 28 (45.9%) 16 (55.2%) 17 (63%) 10 (52.6%)
0.507
1 33 (54.1%) 13 (44.8%) 10 (37%) 9 (47.4%)
Stage III 19 (31.1%) 8 (27.6%) 7 (25.9%) 8 (42.1%)
0.664
IV 42 (68.9%) 21 (72.4%) 20 (74.1) 11 (57.9%)
a Out of the 147 KRAS codon 12 mutant patients, in 11 KRAS codon 12 mutant cases the exact nucleotide change was not identifiable;
b Mean age was 58.8 years (range, 39-78; SD=8.16) for the entire KRAS codon 12 mutant group, 58.1 years (range, 39-76; SD=8.00) for the G12C patients, 59.5 years (range, 41-76; SD=8.14) for the G12V patients, 59.1 years (range, 39-75; SD=8.28) for the G12D patients, and 59.6 years (range, 40-78; SD=8.68) for patients with rare KRAS codon 12 mutations; Data shown in parentheses are column percentages; ECOG PS, Eastern Cooperative Oncology Group performance status.
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4.2.1. Smoking and KRAS and EGFR mutation status
In cohort #1, smoking status and KRAS mutational status did not show a significant correlation (P=0.059; Figure 9A). However, when KRAS mutant cases were combined (all KRAS WT patients vs. codon 12 plus codon 13 KRAS mutants; Table 3) the tendency towards an increased frequency of KRAS mutations in ever-smoker patients reached a statistically significant level (P=0.0189; vs. never-smokers; Chi-square test).
Accordingly, we found a significantly elevated risk for ever-smoker advanced lung adenocarcinoma patients to carry a KRAS mutation (RR=1.93; CI=1.1136-3.3512;
P=0.0089) that translates to an almost two-fold risk of having a KRAS mutant tumor.
In cohort #2, KRAS mutant cases significantly associated with smoking status when compared to the double WT patient population (P<0.01; Figure 9B). Classic EGFR mutation was significantly associated with never-smoker status when compared to all other mutational statuses (Figure 9B; P<0.0001). Next, we investigated the clinical relevance of subtype-specific EGFR and KRAS mutations. We found that rare EGFR mutations are associated with smoking (vs. classic EGFR mutations; Figure 9B;
P=0.0062).
Next, in cohort #1, we investigated the characteristics of patients with KRAS mutations in codon 12 and performed a statistical analysis on their association with amino acid-specific mutational status. Similar to the overall cohort, smoking status and acid-specific KRAS codon 12 mutations showed an almost significant correlation (P=0.055, Table 3).
Therefore, the correlation of mutational status and smoking status was further analyzed (Figure 9B). Codon 12 KRAS mutations were significantly more frequent in current and/or former smokers than in never-smokers (P=0.032, Figure 9B). Importantly, the amino acid-specific mutation subtype analysis identified G12V KRAS mutation as more frequent in never-smokers than among former and current (or ever) -smokers (Figure 9C)
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Figure 9. Distribution of patients according to driver oncogenic mutations and smoking status. (A) In cohort #2, rare EGFR mutations - in contrast to classic EGFR mutations - were significantly associated with smoking (P=0.0062). In cohort #1, (B) KRAS wild-type (WT), KRAS codon 12 and codon 13 mutants and (C) codon 12 subtype-specific KRAS mutants were analyzed. KRAS mutation is significantly more frequent among former or current than in never-smokers (P=0.032, Chi-square test). G12V KRAS mutation is more frequent in never-smokers.
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4.2.2. Patient characteristics and metastatic pattern
Clinicopathological characteristics and KRAS mutational status of patients with different metastatic pattern are shown in Table 6 and Table 7. Among the 903 consecutive lung adenocarcinoma patients identified, 256 (28%) were KRAS mutant and 647 (72%) were KRAS WT. Four hundred three patients presented with non-metastatic disease and 500 cases were metastatic at the time of diagnosis. We found 362 (72%) single-organ and 138 (28%) multiple-organ metastatic cases (Table 6). The most frequent metastatic sites included lung (45.6%), bone (26.2%), adrenal gland (17.4%), brain (16.8%), pleura (15.6%), and liver (11%).
We did not found significant differences in age in the metastatic (61.9±9.4) vs. non-metastatic (61.8±8.9) cohorts or patients with single-organ (62.33±9.3) vs. multiple-organ (60.8±9.7) metastases. Patients presented with only pleural spread (66.8±10.4) were significantly older than those with only lung (62±8.9), bone (60±10.7), adrenal (63.1±6.8), or brain (59.7±9.2) metastases (P=0.0024, P=0.0008, P=0.0132, P=0.002).
Patients with brain metastases were significantly younger than those with lung spread (P=0.0094).
Only in the bone metastatic group we found a higher percentage of male patient when compared to females in adrenal, brain or lung group (56% vs. 49%, 43%, and 45%, respectively, P=0.0479). The proportion of ECOG PS 0-1 was similar in the different organ-specific metastatic subgroups. The proportion of never-smokers was significantly increased in patients with pleural metastases (27%) when compared to all other sites (12.2%, P=0.0018).
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Table 6.Correlation of clinicopathological features, KRAS mutation status and metastatic pattern in the combined cohort at the time of diagnosis in patients with advanced pulmonary adenocarcinoma (n=903).
Metastatic pattern Multiple-organ Single-organ Non-metastatic
Total 138 362 403
Age (mean±SD) 60.8±8.7 62.4±9.3 61.8±8.9
Gender
Male 64 (46%) 181 (50%) 190 (49%)
Female 74 (54%) 181 (50%) 213 (51%)
ECOG PS
0-1 124 (92%) 335 (94%) 382 (96%)
>1 11 (8%) 21 (6%) 15 (4%)
Unknown data 3 6 6
Smoking status
Never-smoker 15 (12%) 52 (16%) 66 (17%) Former smoker 37 (30%) 104 (31%) 115 (30%) Current smoker 71 (58%) 179 (53%) 203 (53%)
Unknown data 15 27 19
KRAS
Wild-type 94 (68%) 263 (73%) 290 (72%)
Mutation 44 (32%) 99 (27%) 113 (28%)
Data shown in parentheses are column percentages.
Metastatic pattern was evaluated at the time of diagnosis. ECOG PS, Eastern Cooperative Oncology Group performance status.
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Table 7.Clinicopathological features, KRAS mutation status and site specific metastatic pattern in the combined cohort at the time of diagnosis in patients with advanced lung adenocarcinoma (n=500*).
Metastatic site Lung Bone Adrenal Brain Pleura Liver
Total 228 131 87 84 78 55
Data shown in parentheses are column percentages.
*The number of site-specific metastatic cases included single and multiple organ metastatic patients at the time of diagnosis. ECOG PS, Eastern Cooperative Oncology Group performance status.
4.2.3. Metastatic site-specific variation of KRAS status
Metastatic site-specific variation of KRAS status is shown in Figure 10. There was no difference in the KRAS mutation incidence between the metastatic (28.6%) and non-metastatic cases (28%) (Table 6, Figure 10A). Patients with multiple-organ metastases showed a non-significant increase in the percentage of KRAS mutation (vs single-organ spread 32% vs 27%, Table 7, Figure 10B).
Importantly, patients with brain (29%), bone (28%) or adrenal gland (33%) metastases demonstrated similar KRAS mutation frequencies (Figure 10C). However, pulmonary metastatic cases demonstrated increased KRAS mutation frequency when compared to
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those with extrapulmonary metastases (35% and 26.5%, P=0.0125, Figure 10C). In contrast, pleural dissemination and liver metastasis associated with decreased KRAS mutation incidence (vs all other metastatic sites; 17% (P<0.001) and 16% (P=0.0023), respectively).
Figure 10. Metastatic site-specific variation of KRAS status. (A) Non-metastatic or metastatic patients (28% vs. 28.6%, ns, Chi-square test), and (B) patients with multiple-organ metastases showed a non-significant increase in the percentage of KRAS mutant cases (vs. single-organ spread, 32% vs. 27%). (C) In the organ-specific analysis, patients with brain (29%), bone (28%) or adrenal gland (33%) metastases demonstrated similar KRAS mutation frequencies. However, pulmonary metastatic cases demonstrated increased KRAS mutation frequency when compared to those with extrapulmonary metastases (35% vs. 26.5%, P=0.0125). In contrast, pleural dissemination and liver metastasis associated with decreased KRAS mutation incidence (17% (P<0.001) and 16%
(P=0.0023), respectively). WT, wild-type; MUT, mutant; Single, single-organ; Multiple, multiple-organ metastasis.
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